In clinical routine blood is the most important source of sample to be analyzed. Though whole blood is the first sample obtained, the whole blood sample usually has to be further processed in order to allow for convenient sample handling or for reliable analyte detection.
The more constituents are present in a sample the more difficult is the analysis of a target analyte comprised therein. Red blood cells contain a dramatic amount of proteins and small molecular weight constituents that potentially interfere with any analyte to be detected. This is one of the major reasons why in clinical routine preferably blood plasma (often simply referred to as plasma, i.e., an anticoagulated whole blood sample; deprived of cells and erythrocytes) or blood serum (often simply referred to as serum, i.e., coagulated whole blood; deprived of cells, erythrocytes and most proteins of the coagulation system, especially of fibrin/fibrinogen), respectively, are used. Whole blood samples also tend to be more difficult to handle, e.g., as compared to serum or plasma. Whole blood tends to be less stable and slow rupture of erythrocytes impairs a reliable measurement of quite a few analytes of interest. In addition, transport and storage of a whole blood sample requires special measures of precaution.
In case an analyte has to be measured from whole blood, it is general practice to collect the whole blood sample and to treat such sample during or immediately after collection of blood with an appropriate anti-coagulant. In clinical routine tubes prefilled with an appropriate anti-coagulant are used for collection of whole blood samples. As the name tells these anti-coagulants block the activation of the coagulation system. Blood cells and erythrocytes shall remain intact as much and as long as possible. The anti-coagulated blood has to be handled very carefully in order to avoid problems, e.g., caused by sedimentation of blood cells or erythrocytes or caused by lysis of erythrocytes. Usually aliquots of such anti-coagulated whole blood sample are then used in the detection of an analyte of interest, e.g., of an analyte that is at least partially comprised within red blood cells.
In addition, at this point in time it does not appear to be feasible to use a whole blood sample in any of the existing online detection methods. It is for example not possible to use a whole blood sample in a clinical diagnostic routine procedure requiring a separation step based on liquid chromatography (LC). Routine liquid chromatographic separation usually is based on a column essentially consisting of a filter unit or frit to protect the column material and the column material required for the separation of the analyte(s) of interest. If whole blood is applied to such column, the column will be blocked rather soon or even immediately, depending on column size and system. This problem makes it merely impossible to use whole blood in an online detection process in combination with an LC-method as for example preferred in clinical routine diagnosis. At present it appears that appropriate separation/handling of a blood sample, e.g., by centrifugation, filtration, precipitation or analyte extraction is essential, before such processed sample can be properly and reliably analyzed.
As indicated above, serum or plasma may be obtained from whole blood and used in the detection of an analyte. Cells and erythrocytes in theory may also be removed by filtration or centrifugation from whole blood. However, these methods are neither appropriate for use in a routine diagnostic setting, nor would they allow for a correct measurement of those analytes at least partially present inside red blood cells.
In a further way of sample processing the analyte of interest is first separated from the majority of potentially interfering substances by selective precipitation or extraction methods. Extraction can be performed in liquid phase or on a solid phase. This shall be exemplified by illustrating some of the procedures used in the detection of immunosuppressive drugs.
Well-known immunosuppressive drugs are, e.g., mycophenolate mofetil (MMF), rapamycin (RAPA also known as sirolimus) and tacrolimus (FK-506). Therapeutic drug monitoring for immunosuppressive drugs is especially important for transplant patients as well as for patients suffering from AIDS (cf., e.g.: Drug. Ther. Perspect 17 (2001) 8-12). Most patients who undergo solid organ transplantation require lifelong immunosuppressive therapy to prevent allograft rejection. But, because many immunosuppressive agents have narrow therapeutic ranges also referred to as therapeutic window, and are associated with various toxicities and the potential for drug interactions, the use of therapeutic drug monitoring (TDM) in conjunction with clinical assessment of patients may be particularly important.
Mycophenolate mofetil is a prodrug. After oral administration, mycophenolate mofetil (MMF) undergoes rapid hydrolysis in the intestine and blood to form its active metabolite mycophenolic acid (MPA). MMF is widely available and is approved in the US and UK for the prevention of renal, hepatic or cardiac allograft rejection in combination with corticosteroids and cyclosporin. The drug has demonstrated superiority over azathioprine in reducing the incidence of acute rejection of renal allografts. The therapeutic trough concentration is in the range of 1-3.5 mg/L. MMF can be measured from plasma and from whole blood.
Tacrolimus is a macrolide antibiotic that was first approved by the US Food and Drug Administration (FDA) in 1994 for the prevention of liver allograft rejection. It is up to 100 times more potent than cyclosporin in vitro, and clinically, it is associated with a greater reduction in the incidence of tissue rejection. Tacrolimus has demonstrated efficacy both as primary immunosuppressive therapy in patients undergoing various transplantation procedures and as rescue therapy for patients with refractory acute allograft rejection after liver or kidney transplantation. The therapeutic trough concentration is in the range of 5-20 μg/L.
Since at least part of the tacrolimus present in the circulation is compartmented within erythrocytes, a whole blood sample is used in the clinical routine measurement of this drug. Tacrolimus can, e.g., be detected by high performance liquid chromatography (HPLC), HPLC mass spectrometry (MS), radio receptor assay (RRA), or by an immunoassay (IA). The latter two methodologies do not detect tacrolimus and certain of its various metabolites with the same sensitivity. This may lead to an interference in the procedure used (Murthy, J. N., et al., Clin. Biochem. 31 (1998) 613-617). At least in the detection of the various tacrolimus metabolites the HPLC-MS-procedure may be considered the gold standard. All the procedures mentioned above, however, require the extraction of tacrolimus from whole blood. Usually acetonitrile is used in clinical routine for the extraction of tacrolimus from whole blood and no method appears to exist that would allow for an online measurement of tacrolimus from a whole blood sample.
Sirolimus is, like tacrolimus, a macrolide antibiotic. It was first approved in 1999 by the US FDA for the prevention of allograft rejection after kidney transplantation, and indeed has shown promising results in this respect when used acutely in combination with cyclosporin and corticosteroids. In vitro, sirolimus is up to 100 times more potent than cyclosporin, and clinically, it may exhibit synergism with cyclosporin, perhaps permitting a reduction in cyclosporin dosage. The therapeutic trough concentration is in the range of 5-15 μg/L.
As for tacrolimus, a significant amount of sirolimus is present within erythrocytes. Therefore extraction of a whole blood sample is required no matter which detection method is used. In clinical routine a sample suspected to comprise sirolimus is subjected to HPLC and sirolimus is detected by ultraviolet light (UV) or by MS/MS. Recently also a microparticle enzyme immunoassay has been described (Jones, K., et al., Clinical Therapeutics 22, Suppl. B (2000) B49-B61).
Folate is the collective name of a group of related molecules differing in oxidation state. Folates are part of the water-soluble vitamin B group and are important as coenzymes for homocysteine metabolism and in the transfer of one-carbon groups required for DNA replication. Inadequate folate status is related to increased risk of neural tube defects, is associated with cardiovascular disease, anemia, with certain cancers and with Alzheimer's disease. Serum or plasma folate concentrations reflect recent dietary intake, whereas erythrocyte folate concentrations are more indicative of body stores (Gunter, E. W., et al., Clin. Chem. 42 (1996) 1689-1694; Fazili, Z., et al., Clin. Chem. 51 (2005) 2318-2325; Pfeiffer, C. M., et al., Clin. Chem. 50 (2004) 423-432). Erythrocyte total folate (red blood cell folate=RBC-folate) is the best measure of whole body folate status. Recent studies have shown that 5-methyl tetrahydrofolate is the dominant folate vitamer in circulating erythrocytes. For the diagnosis of folate deficiency it is recommended that determinations are performed not only from serum or from plasma but also from erythrocytes, since folate is localized to more than 95% in the latter. The concentration in the erythrocytes more truly reflects the actual folate status.
A number of methods are available to measure folate in different matrices. The major analytical methods are microbiological assay, radio immuno assay, chemiluminescence, chromatographic methods and mass spectrometric methods. Most methods are based on competitive binding of folate to folate binding protein.
For the measurement of RBC-folate the use of a hemolyzing reagent is obviously mandatory. For example the ELECSYS assay (Roche Diagnostics GmbH) for determination of RBC folate uses ascorbic acid as lysis reagent. ELECSYS RBC-folate hemolyzing reagent is used together with the ELECSYS folate assay for the quantitative determination of folate in erythrocytes (RBC-folate). Whole blood treated with anticoagulants (heparin or EDTA) is diluted with ascorbic acid solution (0.2%) and incubated for approximately 90 minutes at 20-25° C. Lysis of the erythrocytes takes place, with liberation of the intracellular folate. The hemolysate is then used as a “prediluted” sample (in analogy to serum) for subsequent measurement in the ELECSYS folate assay. The hematocrit value determined in whole blood and the dilution effect brought about by pretreatment of the sample is compensated for in the calculation of the erythrocyte folate concentration (Greiling, H., Gressner, A. M., Lehrbuch der Klinischen Chemie und Pathobiochemie, 3rd ed., Stuttgart, N.Y., Schattauer (1995) pp. 460-462; Gunter, E. W., et al., Clin. Chem. 42 (1996) 1689-1694).
The hemolysate generated by treatment with ascorbic acid can not be used for routine chromatographic procedures. For use of such hemolysate in chromatographic procedure or mass spectrometric determination it is necessary to remove cell debris and precipitated protein prior to analysis.
Debris and precipitated proteins usually are removed from a sample by centrifugation, offline filtration or solid phase extraction.
Solid phase extraction (SPE) is a chromatographic technique which is widely used, e.g., for preconcentration and cleanup of analytical samples, for purification of various chemicals, and for removal of toxic or valuable substances from aqueous solutions. SPE is usually performed using a column or cartridge containing an appropriate resin. SPE procedures have been developed using sorbents which can interact with analytes by hydrophobic, ion exchange, chelation, sorption, and other mechanisms, to bind and remove the analytes from fluids. Since different SPE applications for different classes of analytes can require different sorbents, there is a concomitant need for sorbents with specific properties which have unique selectivity for the analyte or class of analytes of interest, Representative examples of SPE materials and SPE columns, respectively, can be found in U.S. Pat. No. 6,322,695 and U.S. Pat. No. 6,723,236.
Alike to quite a few other analytes of interest, there appears to be no method available that would allow for the detection of sirolimus or tacrolimus in an online procedure from a whole blood sample.
The concentration of hemoglobin itself as well as the ratio of glycated hemoglobin (HbA1c) to non-glycated hemoglobin are important analytes in hematology and diabetes. In such assessment the erythrocytes comprised in a whole blood sample are lysed and the hemoglobin is then measured. U.S. Pat. No. 6,050,956 describes a hemolyzing tube that is prefilled with a standardized amount of a blood dissolving liquid. However, whole blood is first collected into a routine blood collection tube. Thereafter blood is diluted 1 plus 100 into the hemolyzing tube. Due to the very high concentration of hemoglobin a 1 plus 100 dilution of whole blood is possible and no differential hemolysis, i.e., no hemolysis avoiding negative side effects like protein precipitation and/or release of DNA, is required
Various patent families to Coulter International Inc., like U.S. Pat. No. 5,874,310; EP 1 000 356; EP 0 874 988; EP 0 305 491 or EP 0 185 048 relate to the field of hematology and especially to the analysis of blood cells. EP 1 000 356, e.g., describes an improved diluent for dilution of a blood sample that is suited for enumeration and sizing of blood cells, determination of hemoglobin parameters and differentiation of leukocyte sub-populations in a single blood sample. Analysis is performed by use of suitable electronic instrumentation. For such analysis blood is usually collected by a physician, then has to be transported to the clinical laboratory, and only shortly before analysis a lysis reagent is added.
Obviously careful transport of an anti-coagulated whole blood sample is crucial. Freezing and elevated temperature must be avoided. There also is a significant biohazard associated to the transport of an anti-coagulated whole blood sample. A tube that leaks or breaks during transport may contaminate packaging material or might be infectious.
It becomes obvious from the above discussion of the state of the art that whole blood still is a stepchild in clinical routine. All routine procedures even today appear to require an anti-coagulation treatment, high dilution of the sample, and/or the separation or fractionation of an analyte of interest or of a certain class of compounds from the rest of compounds comprised in such sample. In addition, no method for an online measurement of a whole blood sample appears to be available.
It would, however, be highly desirable if whole blood could be used directly and easily as a sample. This would be especially advantageous in an online detection procedure making use of a liquid chromatographic (LC) separation step. It is also obvious that the direct processing of a whole blood sample rendering the processed sample more easy to store, handle and transport would represent an important progress for clinical routine diagnostic applications.
It has now surprisingly been found and could be established that it is possible with great advantages to collect a sample of whole blood into a ready-to-use and single use whole blood sampling tube that is prefilled with a reagent for differential hemolysis of said whole blood sample. The sampling tube according to the present invention greatly facilitates the use of a whole blood sample, renders the handling of such sample and also the transport of such sample easy and convenient, and allows for the direct detection of analytes from a whole blood sample. The collection of whole blood in a sampling tube according to the present invention, e.g., renders whole blood an appropriate sample for direct separation by chromatography and analyte detection, e.g., by mass spectroscopy. This is especially valuable for an analyte that is also present to a relevant extend inside red blood cells, like folate or the immunosuppressive drugs sirolimus and tacrolimus.